The unit has studied the expression of short interspersed repetitive sequence genes of unknown function, into RNA and ribonucleoprotein particles (RNP). 1. We have pursued structural studies of various B1-Alu RNAs. A method was developed to screen genbank for B1 sequences that have the potential to form secondary structures equivalent to the Alu domain found in both the SRP and AFP-derived B1 RNAs. In vivo expressional analysis of some of these sequences has shed light on requirements for in vivo processing of B1-Alu primary transcripts. In order to perform experimental determinations of B1-RNA structure, a recombinant plasmid was constructed which allows T7 polymerase-directed in vitro synthesis of RNA corresponding precisely to either the 210 nt primary or 135 nt processed AFP-derived species. This construct can easily be modified to accommodate other naturally occurring B1 genes. 2. A method was developed for the selective isolation and cloning of B1 sequences expressed as small cytoplasmic RNAs in murine tissues and cell lines. The method involved synthesis of cDNA from gel purified small cytoplasmic B1 RNA followed by homopolymeric tailing and PCR amplification. Subcloning into a derivative of the T7 promoter-containing plasmid described above allows the production of RNA which corresponds to naturally occurring small cytoplasmic RNA. This can be used for sequence and structure analysis, and identification of motifs important for proper expression. 3. The B1-Alu RNA previously identified in murine hepatoma cells which is homologous to the processed 135 nt B1 RNA made in microinjected Xenopus oocytes, has been further characterized. Sedimentation and biochemical characteristics provided further insights into the possible role of this RNA in cellular metabolism. 4. The distribution of the polypeptide Be (p63) previously found to be associated with B1-RNA in microinjected oocytes has been extended to Xenopus Tissue Culture (XTC). As was previously found in Hela cells, synthesis of Be was detectable only after significant cellular proliferation. In XTC Be appeared only after synchronized proliferation of previously quiescent cells, indicating that this protein is either stable with low turnover, or expressed in a cell cycle dependent fashion. 5. Construction of plasmids containing 5' deletions of the AFP-derived B1 gene and no RNA pol II promoters has allowed the continued investigation of B1-specific transcription by Pol III.